Physiological substrates and ontogeny-specific expression of the ubiquitin ligases MARCH1 and MARCH8.

MARCH1 and MARCH8 are ubiquitin ligases that control the expression and trafficking of critical immunoreceptors. Understanding of their function is hampered by three major knowledge gaps: (i) it is unclear which cell types utilize these ligases; (ii) their level of redundancy is unknown; and (iii) most of their putative substrates have been described in cell lines, often overexpressing MARCH1 or MARCH8, and it is unclear which substrates are regulated by either ligase in vivo. Here we address these questions by systematically analyzing the immune cell repertoire of MARCH1- or MARCH8-deficient mice, and applying unbiased proteomic profiling of the plasma membrane of primary cells to identify MARCH1 and MARCH8 substrates. Only CD86 and MHC II were unequivocally identified as immunoreceptors regulated by MARCH1 and MARCH8, but each ligase carried out its function in different tissues. MARCH1 regulated MHC II and CD86 in professional and "atypical" antigen presenting cells of hematopoietic origin, including neutrophils, eosinophils and monocytes. MARCH8 only operated in non-hematopoietic cells, such as thymic and alveolar epithelial cells. Our results establish the tissue-specific functions of MARCH1 and MARCH8 in regulation of immune receptor expression and reveal that the range of cells constitutively endowed with antigen-presentation capacity is wider than generally appreciated.

Criteria for dendritic cell receptor selection for efficient antibody-targeted vaccination.

Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently, there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study, we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First, using mixed bone marrow chimeras, we established that Ag-targeted, but not nontargeted, DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next, we analyzed parameters of DEC205 (CD205), Clec9A, CD11c, CD11b, and CD40 endocytosis and obtained quantitative measurements of internalization speed, surface turnover, and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays, we showed that receptor expression level, proportion of surface turnover, or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore, the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast, targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation, respectively. Therefore, receptor expression levels, speed of internalization, and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination.

Enhanced survival of lung tissue-resident memory CD8⁺ T cells during infection with influenza virus due to selective expression of IFITM3.

Infection with influenza virus results in the deposition of anti-influenza CD8(+) resident memory T cells (T(RM) cells) in the lung. As a consequence of their location in the lung mucosal tissue, these cells are exposed to cytopathic pathogens over the life of the organism and may themselves be susceptible to infection. Here we found that lung T(RM) cells selectively maintained expression of the interferon-induced transmembrane protein IFITM3, a protein that confers broad resistance to viral infection. Lung T(RM) cells that lacked IFITM3 expression were more susceptible to infection than were their normal counterparts and were selectively lost during a secondary bout of infection. Thus, lung T(RM) cells were programmed to retain IFITM3 expression, which facilitated their survival and protection from viral infection during subsequent exposures.

Differential expression of pathogen-recognition molecules between dendritic cell subsets revealed by plasma membrane proteomic analysis.

Dendritic cells (DC) are comprised of several subsets with distinct functions, differing in their capacity to respond to pathogen products. To gain novel insights into their pathogen specificity, we compared the protein composition of the plasma membrane of CD8+ and CD8- DC, directly isolated from mouse spleens. Differences in protein expression were determined using semi-quantitative high-resolution mass spectrometry of label-free plasma membrane-enriched fractions. Our comparative proteomic analysis detected over 1500 proteins, revealing broad differences in expression of pathogen receptors, adhesion molecules and T-cell regulatory molecules. Many of these findings were validated using flow cytometry and Western Blot analysis of integral and luminal surface-associated membrane proteins. This analysis provides major advantages over genomic approaches as it directly measures protein expression at a particular location. Our study highlights the diversity of surface protein expression amongst components of the DC network.

alpha1-acid glycoprotein as a putative biomarker for monitoring the development of the type II reactional stage of leprosy.

Leprosy, a spectral disease manifested on the basis of host immune responses, is complicated by its reactional stages, namely type I reversal reaction (RR) and type II erythema nodosum leprosum (ENL). These reactional stages are characterized by uncontrolled and aberrant immune responses. Biomarkers for reactional stages would aid in early diagnosis, efficient treatment, prevention of neurological complications and prediction of predisposition to reactional stages. In this study, comparative analysis of the serum proteome of leprosy patients by two-dimensional electrophoresis (2DE) followed by mass spectrometry showed differential expression of acute-phase protein alpha (1)-acid glycoprotein (AGP; also known as orosomucoid). AGP levels in untreated ENL cases were significantly higher than in lepromatous leprosy (LL; a non-reactional disease stage) (P=0.0126), RR (P=0.0176) and healthy controls (P=0.0030). These data were confirmed using ELISA. The levels of AGP decreased to normal levels after treatment with multidrug therapy and thalidomide (P =0.0167). In a follow-up study, AGP levels, which were high in the untreated ENL stage, decreased significantly at 5 days ( P=0.0084) and 21 days (P=0.0027) post-treatment. A stage-dependent increase in AGP in an LL patient who progressed into the ENL stage was also shown. Glycosylation analysis by 2DE showed differential expression of acidic glycoforms of AGP in untreated ENL cases. Changes in AGP concentration and differential expression of isoforms correlated with the inflammatory condition in ENL and also with the treatment regimen. Thus, initial validation of AGP as an ENL-specific biomarker and treatment indicator was shown in this study.

Serum proteome of leprosy patients undergoing erythema nodosum leprosum reaction: regulation of expression of the isoforms of haptoglobin.

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of alpha2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition.

Cloning and identification of EDD gene from ultraviolet-irradiated HaCaT cells.

Ultraviolet (UV) radiation is one of the most important external stimuli that affects skin by inducing cancer, inflammation and cell death. To identify the regulation of genes regulated by UV during transformation, normal human keratinocyte cell line, HaCaT, was exposed to multiple doses of UVA+B (UVA - 150-200 mJ/cm2 and UVB - 15-20 mJ/cm2 x 6). Malignant transformation was confirmed by formation of colonies on soft agar and DNA methylation assay. To identify the genes involved in this process, random amplification of polymorphic DNA using RNA from unexposed and multiple exposed cells was performed after each exposure. A few up-regulated genes were identified, cloned and sequenced. One of the genes had homology to EDD (E3 identified by differential display) that was up-regulated at second exposure but was down-regulated in colony-forming cells (cells that received six or more exposures) as determined by RT-PCR. This is a progesterone-induced gene and progesterone treatment reduced the extent of colony formation on soft agar plate. It is possible that hormone therapy may have some effects on skin cancer in vivo.

UV induced bystander signaling leading to apoptosis.

Human keratinocytes (HaCaT) were exposed to UV (A+B) (UVA-350-400 mJ/cm2 and UVB-30 mJ/cm2) which induces apoptosis as evidenced by MTT assay, DNA laddering, Bax and Fas up-regulation. UV induced apoptotic conditioned media (6 h or earlier) did not cause apoptosis in unexposed cells. However, treatment with conditioned medium collected post UV exposure (1 h) induced Bax in unexposed cells as observed by RT-PCR. The induction of cell death was initiated by conditioned medium collected 12 h after UV exposure and the extent of death was increased progressively when conditioned medium collected 24 or 72 h post UV exposure was used. Medium collected 24 h after UV exposure also increased mitochondrial membrane permeability as determined by rhodamine uptake. Conditioned medium induced apoptosis did not involve reactive oxygen species (ROS) unlike UV induced apoptosis indicating that the apoptosis pathway could be different. Interestingly, at high dilution apototic conditioned medium did not induce apoptosis but actually protected cells from UV insult. The role of nerve growth factor (NGF) in UV induced bystander effects are also discussed.

Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase.

The normal human keratinocyte cell line, HaCaT, was transformed using multiple doses of ultraviolet (UV)A+B (UVA, 150-200 mJ/cm(2) and UVB, 15-20 mJ/cm(2) x 6). Malignant transformation was confirmed by upregulation of Cyclin D1 (mRNA) and formation of colonies on soft agar. To identify the genes involved in this transformation process, we have done rapid amplification of polymorphic DNA using RNA from unexposed and multiple-exposed cells. Six percent PAGE showed several differentially regulated genes in exposed cells compared with unexposed cells. Total 19 genes were identified, cloned and sequenced. Three of these 19 cloned genes showed 99% homology at both DNA and protein levels to a stretch of 540 bp (180 aa) of long interspersed element (LINE)-1 reverse transcriptase (RT) open reading frame (ORF-2). Colonies from soft agar showed upregulation of this gene compared with non-colonized (lawn on soft agar) cells as detected by RT-PCR. This data implicates LINE-1 RT (ORF-2) in UV-induced malignancy and can possibly be used as a marker for the diagnosis of UV-induced skin cancer.

Cloning and identification of two unique genes involved in UV induced apoptosis on human keratinocyte (HaCaT) cell line.

Differential gene regulation during UVB induced apoptosis of human keratinocyte cell line (HaCaT) has been investigated. Rapid amplification of polymorphic DNA (RAPD)-PCR was done to identify novel/unique genes in the purified apoptotic and non-apoptotic populations. Two genes were identified and cloned in pGemT vector. One of these genes (apgene-1) was upregulated in UV induced apoptotic cells and in the non apoptotic cells exposed to UV. The other gene (apgene-2) was not detected in apoptotic cells but expressed in non-apoptotic/non necrotic cells that had been exposed to UV. The presence of apgene-1 mRNA was not detected in camptothecin induced apoptotic as well as non apoptotic cells. Apgene-2 was not detected in camptothecin induced apoptotic cells but expressed in non-apoptotic/non necrotic cells. This data indicates differential regulation of these two genes during UV and chemical induced apoptosis in human keratinocytes. Additionally, since apgene-2 was upregulated in the non necrotic/non apoptotic population could be involved in protection.